Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples

Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples Laura Valentina Laverde-Arias, Yeirme Yaneth Jaimes-Suárez, Adriana González-Almario This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6567972/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Persea americana and Theobroma cacao plants can be infected by species of the genus Lasiodiplodia , which cause the dieback disease, and T. cacao can also be infected by Ceratocystis sp., which causes cocoa wilt. These fungi begin by necrotizing the internal vascular tissues and thus, symptoms are not observed in the early stages of infection, making the diagnosis difficult. Therefore, in this study we developed a nested PCR for the early detection of Ceratocystis sp. and Lasiodiplodia sp. from infected and asymptomatic seedling stem samples. A pre-treatment with glycerol and 96% ethanol to soften lignified samples from cocoa and avocado seedlings was standardized and facilitated facilitate DNA isolation from the samples. Then, the amplification of the actin gene from the pre-treated stems confirmed that nucleic acids were free of PCR inhibitors to apply the diagnosis technique and avoid false negative results. The Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R primer sets designed in this study demonstrated to be specific for the detection of Ceratocystis sp. and Lasiodiplodia sp., respectively, with a sensitivity ranging from 2–4 ng/µL. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp., and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp. in a nested-PCR, both pathogens were successfully detected directly from infected stem samples without a fungal isolation step in culture media. The results were easily interpreted by agarose electrophoresis based on fragment size differences, since a 279 bp amplicon was obtained in samples artificially infected with C. cacaofunesta and a 397 bp amplicon observed in samples with L. theobromae and L. subglobosa . In conclusion, the use of the nested PCR protocol standardized in this study with the primers sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp., and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp., allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado seedling stem samples. stem pre-treatment sensitivity asymptomatic Full Text Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 24 Jul, 2025 Reviewers invited by journal 21 Jul, 2025 Editor assigned by journal 14 Jul, 2025 First submitted to journal 05 Jul, 2025 Editorial decision: Major revisions 21 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6567972","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488611257,"identity":"77a1c7d5-b0f0-4735-96ba-ebac00a66401","order_by":0,"name":"Laura Valentina Laverde-Arias","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACPnYwxczAwN4ApAuA+AABLWzMMC08IKUGJGmRSCBaC/MziY97rOX4Z74xe/DDgEGO70YC44cPeLWwmUnOeJZuLHE7x9ywx4DBWPJGArPkDPwOMzbmOXA4seF2jpkEjwFD4oYbCWzMPHi1sH82/nPgcP38m2fMJP8YMNQToYXH8DHDgcMJBjd4zKSBtgAZhLUUPuw5kG648UxambSMgYThzDMPm/H6hZ+9fcOBHwes5eWOH94m+abCRp7vePJBvCGGDiSAmLGBBA2jYBSMglEwCrABAN7ORQCfo71bAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0003-0669-6686","institution":"Universidad Nacional de Colombia - Sede Bogotá: Universidad Nacional de Colombia","correspondingAuthor":true,"prefix":"","firstName":"Laura","middleName":"Valentina","lastName":"Laverde-Arias","suffix":""},{"id":488611258,"identity":"6fc57bac-2813-4527-90bf-2e130c9509c7","order_by":1,"name":"Yeirme Yaneth Jaimes-Suárez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yeirme","middleName":"Yaneth","lastName":"Jaimes-Suárez","suffix":""},{"id":488611259,"identity":"5f14021b-2a72-47b9-9a60-946907b62a02","order_by":2,"name":"Adriana González-Almario","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"","lastName":"González-Almario","suffix":""}],"badges":[],"createdAt":"2025-04-30 21:44:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6567972/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6567972/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87434775,"identity":"a9344ea0-9a8c-4721-bfe1-2a2a38d82a3a","added_by":"auto","created_at":"2025-07-23 18:27:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":550156,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptFinalTPP.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6567972/v1_covered_35617c70-e48d-4b2b-8bc4-bb0332440db5.pdf"}],"financialInterests":"","formattedTitle":"Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"stem, pre-treatment, sensitivity, asymptomatic","lastPublishedDoi":"10.21203/rs.3.rs-6567972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6567972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePersea americana\u003c/em\u003e and \u003cem\u003eTheobroma cacao\u003c/em\u003e plants can be infected by species of the genus \u003cem\u003eLasiodiplodia\u003c/em\u003e, which cause the dieback disease, and \u003cem\u003eT. cacao\u003c/em\u003e can also be infected by \u003cem\u003eCeratocystis\u003c/em\u003e sp., which causes cocoa wilt. These fungi begin by necrotizing the internal vascular tissues and thus, symptoms are not observed in the early stages of infection, making the diagnosis difficult. Therefore, in this study we developed a nested PCR for the early detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. from infected and asymptomatic seedling stem samples. A pre-treatment with glycerol and 96% ethanol to soften lignified samples from cocoa and avocado seedlings was standardized and facilitated facilitate DNA isolation from the samples. Then, the amplification of the \u003cem\u003eactin\u003c/em\u003e gene from the pre-treated stems confirmed that nucleic acids were free of PCR inhibitors to apply the diagnosis technique and avoid false negative results. The Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R primer sets designed in this study demonstrated to be specific for the detection of \u003cem\u003eCeratocystis\u003c/em\u003e sp. and \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., respectively, with a sensitivity ranging from 2\u0026ndash;4 ng/\u0026micro;L. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for \u003cem\u003eCeratocystis\u003c/em\u003e sp., and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp. in a nested-PCR, both pathogens were successfully detected directly from infected stem samples without a fungal isolation step in culture media. The results were easily interpreted by agarose electrophoresis based on fragment size differences, since a 279 bp amplicon was obtained in samples artificially infected with \u003cem\u003eC. cacaofunesta\u003c/em\u003e and a 397 bp amplicon observed in samples with \u003cem\u003eL. theobromae\u003c/em\u003e and \u003cem\u003eL. subglobosa\u003c/em\u003e. In conclusion, the use of the nested PCR protocol standardized in this study with the primers sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for \u003cem\u003eCeratocystis\u003c/em\u003e sp., and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for \u003cem\u003eLasiodiplodia\u003c/em\u003e sp., allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado seedling stem samples.\u003c/p\u003e","manuscriptTitle":"Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 17:55:30","doi":"10.21203/rs.3.rs-6567972/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-24T13:01:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-21T13:46:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-14T14:43:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Plant Pathology","date":"2025-07-06T01:00:38+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2025-05-21T13:49:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"012f7892-628f-479f-99b3-2b440898b463","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-06T19:18:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 17:55:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6567972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6567972","identity":"rs-6567972","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}